An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing

Monosynaptic viral tracers are essential tools for dissecting neuronal connectomes and for targeted delivery of molecular sensors and effectors. Viral toxicity and complex multi-injection protocols are major limiting application barriers. To overcome these barriers, we developed an anterograde monosynaptic H129Amp tracer system based on HSV-1 strain H129. The H129Amp tracer system consists of two components: an H129-dTK-T2-pacFlox helper which assists H129Amp tracer’s propagation and transneuronal monosynaptic transmission. The shared viral features of tracer/helper allow for simultaneous single-injection and subsequent high expression efficiency from multiple-copy of expression cassettes in H129Amp tracer. These improvements of H129Amp tracer system shorten experiment duration from 28-day to 5-day for fast-bright monosynaptic tracing. The lack of toxic viral genes in the H129Amp tracer minimizes toxicity in postsynaptic neurons, thus offering the potential for functional anterograde mapping and long-term tracer delivery of genetic payloads. The H129Amp tracer system is a powerful tracing tool for revealing neuronal connectomes.

Remarks to the Author: Feng Xiong et al present a new experimental approach for anterograde neurotracing with HSV129 vectors that reduces the experimental timeline from several weeks to under one week. The authors claim that their amplicon helper virus system overcomes several of the problems associated with existing approaches of anterograde labeling that utilize HSV namely low labeling intensity and high toxicity in postsynaptic neurons. In general the data do support this claim and demonstrate the potential of the amplicon system. The idea behind this new approach is pretty clever and the amplicon system could have a significant impact in the field even though it does not solve the problem of high toxicity in 1st order neurons. However, it is my understanding that 1st order neurons should be either green or double -labeled as presented in figure 1c. In practice the authors observe numerous 1st order neurons that are labeled brightly red (Fig.2c). This suggests that the system does not always work as it is presented in Figure 1c. The field has seen other ingenious approaches published in high impact journals that looked quite promising but have not found widespread use presumably because of experimental difficulties and problems with reproducibility. To allow a better assessment of the amplicon system in particular with regards to its reproducibility the authors should present a summary of the number and percentage of double labeled cells from several experiments like the one shown in Fig. 2c. I would also like to see some data on the robustness of this system that is how sensitive it is to changes in the Amplicon/Helper ratio.
Reviewer #2: Remarks to the Author: This manuscript describes the development and testing of several new reagents for anterograde trans-neuronal tracing. While the results presented clearly show that the new reagents will have utility for experiments that are not possible with other existing tracers, there are many concerns about various claims that are made without experimental support, and/or potential limitations or labeling artifacts that the authors do not consider. The manuscript would also benefit considerably from clearer descriptions of the various reagents and how they are expected to behave and interact with each other. (The Overview below provides an example of how to do this.) This review begins by providing an overview of the various reagents used and their apparent intended uses. Then the review turns to describing possible limitations to the methods that the authors either fail to consider or claim to have considered but do not support with definitive evidence. The review then turns to problems with organization of the manuscript or lack of detail in descriptions that make it difficult to follow and which could be remedied by some re-writing. Various edits are suggested in the context of the original text. Finally, there are several minor points that could be addressed.

Overview of the reagents described and their intended uses:
The manuscript describes three different tracing systems. All three have in common that they utilize an HSV-H129 strain helper virus to allow the production/packaging of an amplicon vector ("tracer") that then spreads anterogradely to postsynaptic neurons. The helper is called H129-dTK-T2-LoxN-pac-LoxN. dTK refers to deletion of TK from the genome. TK is required for viral replication in dividing cells, such as neurons. T2 refers to the insertion of genes for TdTomato such that infected cells will express TdTomato. LoxN-pac-LoxN refers to the fact that the pac sequence is floxed allowing for its removal following Cre-recombination. This is relevant because pac is required for packaging of viral genomes into viral particles. There are two different tracers used in these experiments as well as two different H129 viruses. How each is used depends on experimental goals of the 3 different systems.
The first system is intended to identify the directly postsynaptic neurons downstream of neurons infected in a brain area of interest. It does not distinguish between outputs of different neuron types in the brain area of interest. Here the helper is H129-dTK-T2-LoxN-pac-LoxN and the tracer is H129ampCTG, which expresses Cre-recombinase, TK, and GFP. These are mixed together and co-injected into the region of interest. In neurons that are co-infected, the helper genome is recombined by Cre in order to remove pac and prevent trans-neuronal spread of the helper. Expression of TK allows the helper to generate all of the required viral structural components and package the tracer genome into amplicons which are then transmitted down the axons and transneuronally to downstream recipients. GFP expression from the tracer then marks the downstream neurons. Insofar as pac is removed from the helper genome, the helper will not spread beyond the initially infected neurons. Furthermore, the tracer is unable to spread beyond the first synaptic step because it is unable to replicate in the absence of helper. The absence of helper from the downstream neurons is also expected to mitigate toxicity. GFP expression from the amplicon tracer is expected to be transient (cite references). If this approach is employed in a cre-reporter mouse line, such as AI14, expression of Cre from the amplicon tracer can permanently label downstream neurons.
The second system is intended to identify the directly postsynaptic neurons downstream of a selected population of neurons that express cre-recombinase (from a mouse driver line) in a brain area of interest. It is intended that the outputs of neighboring cre-negative neurons in the region of interest will not have their outputs labeled (but see below). For these experiments, the same helper is used (H129-dTK-T2-LoxN-pac-LoxN) but the amplicon (H129amp-DIO-TG) expresses only TK and GFP (not Cre), and their expression requires Cre-recombination. When this helper and tracer are co-injected in an area of interest of a cre driver line, pac will be removed from the helper genome in Cre+ cells, preventing it from spreading. Cre will also recombine the amplicon, allowing it to express TK such that the helper can replicate and facilitate production of tracer amplicons that spread to and express GFP in downstream neurons. Cre-negative neurons infected with helper and tracer will initially lack TK and will therefore not be able to produce amplicon tracer. Therefore, the downstream neurons of Cre-neurons would not be labeled. Note however, that for brain regions in which Cre-positive and Cre-negative neurons are interconnected (such as auditory cortex), labeling of downstream neurons will not actually be selective for those connected to the Cre-expressing neurons, as intended. This is because TK expressing amplicons will spread to Cre-negative neurons allowing the helper to be active and produce tracer amplicons in the Crenegative neurons. See further below.
The third system is intended to trace circuits anterogradely across two synaptic steps. It uses the first system described above (H129-dTK-T2-LoxN-pac-LoxN helper and H129ampCTG tracer) to initiate transfer of the tracer to downstream targets. In addition, a second H129-based viral tracer that was described in a previous publication (H129-dgK-G4) is injected into a downstream region. H129-dgK-G4 has an envelope protein required for packaging infectious articles deleted and expresses GFP. It can be transcomplemented by expression of gK to mediate monosynaptic anterograde spread. In order to trace connections across a second synaptic step an AAV helper virus with Cre-dependent gK is injected into the downstream region of interest at the same time that the H129-dTK-T2-LoxN-pac-LoxN helper and H129ampCTG tracer are injected. The H129-dgK-G4 is injected 3 weeks later. In the downstream target region H129ampCTG tracer that has spread to postsynaptic neurons expresses Cre and recombines the AAV helper. The resulting gK expression allows the H129-dgK-G4 to spread an additional synaptic step resulting in GFP label.
Possible limitations and labeling artifacts that are not considered or for which claims against them are not supported: The description above describes how it is hoped that the tracing systems will work. But there are many possible scenarios not considered by the authors or not ruled out by results presented, that would result in very different undesired outcomes. Below, these are each considered in turn with a description of the relevant reagents and experiments and data presented that are relevant. 1) For tracing system 1, it is claimed that helper virus does not spread from the initially infected neurons and that, as a consequence, the spread of tracer is monosynaptic (not multi-synaptic spread). The validity of this claim relies on the unproven assumption that Cre-recombination from the tracer will successfully recombine and remove pac from every copy of the helper genome. This seems unlikely given that the helper will rapidly replicate in the presence of TK expressed from the tracer. To support these claims it is necessary to show that there is no TdTomato expressed at postsynaptic target regions at very short times (1 or 2 days) after injection of the tracer system. For example, following injections of the tracing system into AC, please show photographs of the MG at d1 and d2 post-injection. Are there any red cells observed? Photographs at d5 are not adequate because it is clear that all cells infected with helper would have been killed and eliminated at d5. This is clearly shown in Fig. 2d which illustrates that red fluorescent cells have disappeared from the AC between d1 and d5. (Compare to Fig. 1c.) If there are red cells in postsynaptic targets of the AC at d1, then it is clear that they could mediate spread of tracer another synaptic step. Please show results that clearly illustrate a lack of spread in cases where it might be expected. For example, the results state: "… but not in further downstream second order connected nuclei (data not shown). These results show that H129Amp tracer anterogradely labels directly connected postsynaptic neurons limited to monosynaptic connections." It is not possible to show something with results that are not shown. Please specifically state what structures were observed that might have potentially been labeled by multisynaptic spread. Such structures should contain the GFP-labeled axon terminals of neurons that were trans-neuronally labeled with the tracer. But they should not contain labeled postsynaptic neurons. Please provide photographs illustrating the presence of axon terminals in these locations without postsynaptic labeling.
2) The authors do not consider the known fact that both the helper and the tracer are packaged with envelope proteins that will mediate retrograde infection. The H-129 strain is biased toward anterograde because it is trafficked down the axons of infected neurons. But there is not any mechanism that prevents infection of axon terminals and retrograde spread to the infected cell bodies. As a result, it is expected that injections of the tracing system into cortical locations (such as AC) will result in direct infection of thalamic neurons (such as in MG). This should be clearly visible as TdTomato labeling in the MG following injections of either tracer or helper alone (or both together) into AC. Please show evidence that none of these occurs. For example, show photographs of the MG at day 1 after injections of the tracing system into AC. Are there any red neurons in the MG. Again, note that the absence of red label in MG at day 5 is not sufficient because directly infected neurons are clearly dead and disappear by d5. Please also show evidence that tracer does not retrogradely infect and express GFP in the absence of helper. This is a crucial control. But surprisingly there are no data presented in the manuscript showing the nature of direct infection with the tracer in the absence of helper. Please show photos of the AC and other pre-and postsynaptic targets of AC (e.g. SC, MG, contralateral AC) following injections of tracer without helper.
3) The second "starter-specific" tracing system is unlikely to actually be specific as intended and claimed. This system utilizes a Cre-dependent tracer (H129Amp-DIO-TG) and is intended to trace the monosynaptic outputs selectively from Cre-expressing cells. The authors do not describe the rationale behind how this should work, but apparently they expect that there will not be any expression of TK in cells that do not express Cre. And since Cre-negative cells at the primary injection site are infected with helper but do not express TK, the helper will not replicate in Crenegative cells. It is also assumed that tracer will not spread locally between Cre-positive and Crenegative cells. But these assumptions are certainly wrong for the cortical experiment shown. This is because there are direct connections between the Cre-positive and the Cre-negative cortical cells. It is therefore unlikely that the postsynaptic label observed in distant structures reflects selective outputs from the Cre-positive cortical cells. The expected mechanism for non-selective spread is as follows. Following injections of the tracer and helper into the cortex, both vectors infect both Cre+ and Cre-neurons. In the Cre+ neurons, pac is removed from the helper and the tracer expresses TK and GFP. pac is not removed from the helper that is in the Cre-neurons. Tracer spreads from the Cre+ to connected Cre-neurons where it expresses TK and allows replication of the helper and further production of tracer amplicons in the Cre-negative neurons. This allows the tracer to spread an additional synaptic step and to label the outputs of Creneurons. In addition, helper with intact pac is also replicated and can spread from the Creneurons, resulting in further multisynaptic spread.
The authors do not appear to have considered this and did not use a Cre-expressing mouse line that is capable of demonstrating this artifact. For example, it is expected that if the experiment were conducted in the AC of PV-Cre mice expressing Cre in parvalbumin inhibitory neurons (which make only local cortical connections) "starter-specific" tracing would not result in spread outside the cortex. But the artifact described would allow the spread of tracer from PV interneurons to cortical excitatory neurons, and then from the excitatory cells to postsynaptic neurons in more distant targets. It is necessary to show results from a Cre line in which it is possible for this artifact to be revealed and evaluated. Another such Cre line would be Tlx3-Cre which expresses Cre in cortical layer 5 pyramidal neurons that make cortico-cortical projections but not those that project to subcortical structures. If the spread is actually "starter-specific", use of the system in this line should result in anterograde tracing to cortical targets but no labeling in subcortical structures such as superior colliculus.
Other comments/suggestions: "Although multiple anterograde monosynaptic tracers have been developed based on the herpes simplex virus 1 (HSV-1) strain H129 (H129), functional mapping for output connectome requires further technical development." Suggest changing to "…connectome would benefit from further …".
"Both retrograde and anterograde monosynaptic tracing are achieved by combining two components: i) a viral tracer (tracer), which is a replication-or transmission-deficient virus with one or more genes deleted whose trans-neuronally infects connected neurons requires the assistance of ii) a helper virus (helper) which complimentarily expresses the tracer's deficient gene(s), thus supporting tracer replication and transmission." Suggest the following edits: "Both retrograde and anterograde monosynaptic tracing are achieved by combining two components: i) a conditionally competent viral tracer (tracer) and ii) a helper virus (helper). The viral tracer is replication-and/or transmission deficient due to the deletion of one or more genes that are required for transneuronal spread to connected neurons. The helper virus expresses genes which replace or complement tracer's deficient gene(s), thus supporting tracer replication and/or transmission." The following sentence is factually incorrect and needs to be changed. "The AAV helper has limited expression efficiency due to a single copy of target gene and deficiency in replication, thus needs to be administrated first of two separate injections to allow sufficient target expression and accumulation to support replication and transmission of tracer." AAV genomes concatenate to incorporate many gene copies into episomes. Thus, AAV is not limited to a single copy, and is capable of expressing at very high levels. It is more relevant to this manuscript that expression is slow and that AAV has a limited payload. Suggest changing to: "Due to the slow onset of expression for AAV vectors, the use of AAV necessitates two separate injections, first helper and then tracer, to allow sufficient expression and accumulation to support replication and transmission of the tracer." The introduction provides no context for the statement: "Moreover, for the first time, input-defined postsynaptic neurons' monosynaptic anterograde tracing is succeeded by combining H129Amp tracer system with H129-dgK-G4 tracer." For the reader to understand this statement it will be necessary to describe the H129-dgK-G4 tracer. What are the expected advantages (if any) of using H129-dgK-G4 tracer with H129amp tracer? How is it possible to combine these for cell type specific output labeling since H129-dgK-G4 tracer expresses pac and is not disabled by credependent pac removal?
There is nothing in the Introduction or the Results description of generation, production and tracing principles to make it clear that, for the first tracing system described, the initial infection or production of tracer is not cell type specific. Please state clearly that the initial injection of tracer and helper will infect and spread from all types of neurons at the injection site.
"Abundant AC neurons were labeled by H129Amp tracer (green) and/or helper (red)." Why are neurons labeled red? Is there a red fluorescent protein expressed from the helper genome? Is the tissue stained with an antibody against the helper? There is nothing in the introduction or the results description of generation, production and tracing principles to indicate that TdTomato is expressed from the helper genome. This can only be guessed by looking at Figure 1b. There is also nothing in the figure 1 legend to indicate that this guess is correct. The following edits are suggested. At line 125 rewrite as: "The H129Amp tracer system is composed of H129Amp tracer and H129-dTK-T2-LoxN-pac-LoxN helper. H129-dTK-T2-LoxN-pac-LoxN helper has the thymidine kinase gene deleted (dTK), expresses two copies of the fluorescent protein tdTomato (T2), and its pac gene can be deleted by Cre-recombination (LoxN-pac-LoxN). Due to the identical infection features of H129amp tracer and helper, the system it requires only a single-injection instead of the conventional sequential twice-injection." Also update the figure 1 legend to clearly indicate what is schematized in 1b.
The ED Fig. 2 legend and the figure refer to LP. Based on the photograph it appears that the location indicated by LP is the lateral pulvinar nucleus of the thalamus, which receives direct input from SC. But the figure legend states: "LP, pretectal area;". This is probably wrong.
"These results show that the postsynaptic neurons labeled by H129Amp tracer system maintain normal physiological conditions, thus allowing for functional connection mapping." This result cannot be generalized to all neurons labeled with the tracer. This only applies to cells that are still present after 14 days. Other cells may have died.
"Notably, H129Amp tracer system is also capable of achieving input-defined outputs mapping from specific starter neurons, which cannot be done using transsynaptic AAV2/1." There is no reason to expect that this could not be done with transsynaptic AAV2/1. If AAV2/1 expressing Cre is used in a mouse with Cre-conditional Chr2 expression (either from the genome or from an AAV-DIO-Chr2 injected at the postsynaptic site) then the same experiment could be done.
"Fast tracing. All current monosynaptic tracers, both retrograde and anterograde, use AAVs as helpers." This statement is not true. For example, monosynaptic rabies tracing has been conducted using mouse lines that have cre-dependent or tTA-driven expression of the helper genes. Thus, there is no need for injection of AAV helper or to wait for expression of helper genes.
"In addition to the single-copy target, deficiencies in the replication of viral genome and production of viral particle further limit the expression efficiency of AAV genetic payload." As noted previously, AAV is able to express it's payload very efficiently and without toxicity. Replication is not necessary for efficient long-term expression from AAV vectors. "In the novel H129Amp tracer system, helper, the toxic viral protein producer, is left behind in the starter neurons." As noted above, there are no data presented to support this assertion. It is plausible that the Cre expression from the tracer does not completely remove cap from all helperinfected neurons and that some helper spreads trans-neuronally, along with the tracer. Subsequently, the co-infected trans-neuronally labeled cells die. This is consistent with the observation that GFP expression does not persist in labeled postsynaptic neurons. Please explain why GFP expression is not persistent. I was able to find this in papers cited but readers should not have to track down the relevant facts.
Recent improvements in HSV amplicon vectors might allow more stable gene expression. For example see Soukupova et al., 2021; Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells. https://www.cell.com/molecular-therapyfamily/methods/fulltext/S2329-0501(21)00060-7. This might be worth mentioning in the discussion.
Reviewer #3: Remarks to the Author: In this study, Xiong et al. developed a novel HSV-1-H129 amplicon tracer system as a monosynaptic viral tracer for dissecting neuronal connectomes and targeted delivery of molecular sensors and effectors. The improvements of the H129Amp tracer system shorten the experiment duration from 28-days to 5-days for fast-monosynaptic tracing and minimize toxicity in the postsynaptic neurons. The idea behind this study is very exciting. However, some of the experiment's design and interpretations need more data and or to be further clarified and developed. 1. The authors stated:" For anterograde monosynaptic tracing, the tracer system was administrated into the brain region of interest by a single-injection. In neurons, H129Amp tracer does not replicate alone since it contains no viral gene, but it efficiently expresses Cre, TK, and GFP with its multiunit cassettes in the concatemeric pseudo-genome (Fig. 1d)." It is possible that the GFP can be present in other brain areas to a retrograde transfection mechanism and or transneuronally. 2. The authors should provide data images that include a single injection of H129Amp tracer, showing that the GFP expression is only visible in the injection site and not outside the injection site at the least Day7 and Day 21 post-injection. 3. The same experiments need to be repeated for the helper. 4. Figure 2a and b provided by the authors make it impossible to determine that the virus was injected into the primary AC. 5. Figure 2c. What is the fraction of the co-transfected neurons compared to the single transfected and in which layers of the AC are located? The figure legend of this figure is very confusing. For example, it is not clear if the panels h1 and h2 are a high magnification of the pyramidal neurons of the contralateral AC. 6. The panel 2h depicts the contralateral AC with only a few neurons. Can the authors provide the number of the neurons that are GFP positive in the Cont AC? This is extremely important for the experiments performed in the next section (Figure3). 7. There is no information about the amount of the virus injected into the brain and the stereotaxis coordinates. 8. The authors also indicated that they had determined empirically that after 7 days, the number of the potential co-labeled (potential starter) decreased and dimmed. Can the authors further explain this? Are the neurons dead? 9. The authors stated that:" Many GFP+ neurons were clearly observed in downstream nuclei are directly innervated by AC neurons, including contralateral auditory cortex (Cont. AC), lateral amygdaloid nucleus (LA), medial geniculate nucleus (MG), external globus pallidus (GPe) and locus coeruleus (LC) (Fig. 2e-i), but not in further downstream second-order connected nuclei (data not shown). These results show that H129Amp tracer anterogradely labels directly connected postsynaptic neurons limited to monosynaptic connections." These results are not showing that it is due to monosynaptic connections. 10. Can the authors provide more information about the theRph3a-Cre transgenic mice (i.e., cell type, layers, etc.)? 11. The authors stated:" Next, we mapped the outputs of the input-defined neuron subpopulation anatomically and functionally with H129Amp tracer system. H129Amp tracer system (H129Amp-CTG tracer and helper) was administrated into the AC in the left hemisphere (L-AC) of wildtype C57BL/6 mice as described above, and AAV2/9-DIO-ChR2-mCh was simultaneously injected into the right hemisphere AC (R-AC) in the same mice (Fig. 3a). R-AC receives inputs from the L-AC, and projects back to L-AC, as well as the right hemisphere MG and LA (R-MG and R-LA) (Fig. 3a). Newly propagated H129Amp tracer transmits from the L-AC to the postsynaptic neurons in the R-AC and provides Cre allowing the AAV2/9-DIO-ChR2-mCh to express ChR2-mCherry. As we expected, on Day21 the soma of mCherry labeled neurons (mCh+) were clearly observed in the R-AC (Fig. 3b), but not in any other regions (data not shown)." Can the authors provide a better image of the R-AC (figure 3b)? It is impossible from the actual imagine concluding that it is the R-AC. What is the fraction of neurons that are positive to ChR2? It looks like that there are more ChR2-positive (figure 3b) than GFP-positive (figure 2h) neurons in the contralateral AC. Why? 12. All the Experiment performed in figure 3 and 4 need further experiments before a conclusion like this can be drawn:" Altogether, these results demonstrate that the H129Amp tracer systems (H129Amp-CTG and H129Amp-Flp-DIO-TG tracers) in combination with the appropriate reporter AAVs are capable to not only anatomically but also functionally map the output pathways of subpopulations of input-defined neurons."

Reviewer #1 (Remarks to the Author, italicized):
Feng Xiong et al present a new experimental approach for anterograde neurotracing with HSV129 vectors that reduces the experimental timeline from several weeks to under one week. The authors claim that their amplicon helper virus system overcomes several of the problems associated with existing approaches of anterograde labeling that utilize HSV namely low labeling intensity and high toxicity in postsynaptic neurons. In general the data do support this claim and demonstrate the potential of the amplicon system. The idea behind this new approach is pretty clever and the amplicon system could have a significant impact in the field even though it does not solve the problem of high toxicity in 1st order neurons.
(1) However, it is my understanding that 1st order neurons should be either green or double -labeled as presented in figure 1c. In practice the authors observe numerous 1st order neurons that are labeled brightly red (Fig.2c). This suggests that the system does not always work as it is presented in Figure 1c.

Reply (1):
We thank the reviewer for their acknowledgment of the improvements conferred by our novel system and for allowing us the opportunity to present our new tracer system with greater clarity. H129Amp tracer system consists of two components: the tracer and the helper. After the H129Amp tracer system is simultaneously injected into the brain, the local neurons may be infected by the following potential combination of components: i) tracer alone (green), ii) helper alone (red), and iii) both the tracer and helper (yellow, Reply Fig. 1). The following description takes the H129Amp-CTG tracer as a representative example：i) Neurons that are infected by the tracer alone are labeled green by GFP. The H129Amp tracer, represented by H129Amp-CTG, carries a pseudogenome formed by ~14-copy of concatemeric expression cassette of Cre-TK-GFP (CTG). The H129Amp tracer contains no viral genes (except TK), and cannot replicate in the neurons by itself. But the 14-expression cassettes of CTG simultaneously and constitutively express GFP. Therefore, the cells infected by the tracer alone are labeled green. For these tracer alone infected neurons, there is no virus progeny produced in these neurons and no virus spread occurs. ii) Neurons that are infected by the helper alone are labeled red by tdTomato. The helper H129-dTK-T2-pac Flox is thymidine kinase gene (dTK) deleted, which severely impaired its replication in neurons. The 2copy tdTomato expression cassettes (T2) of the helper consistently express and accumulate the fluorescent protein tdTomato, thus labeling the cells red as shown in manuscript Fig. 2c. Again, for these helper alone infected neurons, there is also no virus progeny produced in these neurons and no virus spread occurs. iii) Neurons that are coinfected by both the tracer and the helper are labeled yellow by both GFP and tdTomato. As described in the manuscript, co-infected tracer and helper obligatorily support each other to produce viral progeny, and the expression of tdTomato and GFP co-labeled the neurons yellow. The progenies of H129Amp-CTG tracer are produced in these starter neurons, and further transmit to the postsynaptic neurons and label them green. Only these co-infected neurons are the potential starter cells for anterograde monosynaptic tracing using the H129Amp tracer system. Figure 2c in the manuscript shows the injection site at Day 1 after the tracer/helper simultaneous injection. The green, red, and yellow cells represent the neurons infected by tracer alone, helper alone, and both tracer and helper, respectively, as described above. The corresponding explanation and description have been added in the revised manuscript (line 141-144).
Reply Fig. 1: Schematic infection and labeling at the tracer system injection site. Elements of this can be found in the revised Fig. 1e in the manuscript.
(2) The field has seen other ingenious approaches published in high impact journals that looked quite promising but have not found widespread use presumably because of experimental difficulties and problems with reproducibility. To allow a better assessment of the amplicon system in particular with regards to its reproducibility the authors should present a summary of the number and percentage of double labeled cells from several experiments like the one shown in Fig. 2c.

Reply (2):
We agree with the reviewer that this is an important piece of information. We performed quantitative analysis of the labeled AC neurons. An average of 588 ± 103 (means ± SEM) yellow neurons (coinfected by both tracer and helper), 352 ± 95 green neurons (infected by tracer alone), and 210 ± 56 red neurons (infected by helper alone) are observed in AC (injection site) (revised Fig. 2c). Double labeled cells account for 51% of total labeled cells, and green and red cells account for 31% and 18%, respectively. The result has been added in the revised manuscript as Fig. 2c, and described in the text accordingly (line 233-236). However we think that it is important to qualify this result by noting that the reproducibility of the co-infection efficiency is dependent on many variables, including the quality of the viral tracer, the ratio of tracer/ helper, and potentially the animal species and the specific circuit to be traced, and perhaps other factors that we have not yet identified. With this cautionary note in mind, we strongly suggest researchers perform test tracing and carefully optimize the component viral titers in relation to tracing quality when they are applying any kind of tracers, see also (PMID: 28499404, 32824837, 33367996, and 35012591). Experimental consistency is an important issue to be concerned about for viral tracer application and was indeed one of our primary motivations to work out novel strategies to develop the present H129Amp tracer system with its streamlined singleinjection protocol rather than the conventional sequential dual-injection protocol.
(3) I would also like to see some data on the robustness of this system that is how sensitive it is to changes in the Amplicon/Helper ratio.

Reply (3):
The reviewer highlights the important issue of different tracer/helper ratios as they impact tracing robustness. In the original manuscript, we depicted our optimized condition with few details on how we arrived at that optization. We now show the conditions that we tested and present these in Supplementary Fig. 1 to give the reader a better sense of how to design similar optization protocols for their own applications.
To optimize tracing outcomes, we tested a range of tracer/helper ratios, including 10:1, 5:1, 1:1, 1:5, and 1:10 ( Supplementary Fig. 1a). H129Amp tracer (represented by H129Amp-CTG) and H129-dTK-T2-pac Flox helper (the only one, shared in all the experiments) were injected into AC of wildtype C57BL/6 mice as the indicated ratio, and the results were observed at Day 5. For tracer/helper ratios of 10:1 and 1:10, no neurons at the postsynaptic nuclei (represented by Cont. AC) were labeled by GFP. When tracer and helper were applied at ratios of 5:1, 1:5, and 1:1, GFPlabeled neurons were observed at Cont. AC, a representative postsynaptic nucleus. Quantification analysis showed that most GFP-labeled neurons were observed in Cont. AC with a 1:1 tracer/helper ratio ( Supplementary Fig. 1b and c). These results suggest that tracer/helper at 1:1 ratio has the best tracing effect, and therefore is applied in all further tracing experiments.
These results have been added to the revised manuscript as Supplementary Fig. 1.

Reviewer #2 (Remarks to the Author):
This

Cre-recombination. This is relevant because pac is required for packaging of viral genomes into viral particles. There are two different tracers used in these experiments as well as two different H129 viruses. How each is used depends on experimental goals of the 3 different
systems.

The second system is intended to identify the directly postsynaptic neurons downstream of a selected population of neurons that express cre-recombinase (from a mouse driver line) in a brain area of interest. It is intended that the outputs of neighboring cre-negative neurons in the region of interest will not have their outputs labeled (but see below). For these experiments, the same helper is used (H129-dTK-T2-LoxN-pac-LoxN) but the amplicon (H129amp-DIO-TG) expresses only TK and GFP (not Cre), and their expression requires Cre-recombination. When this helper and tracer are co-injected in an area of interest of a cre driver line, pac will be removed from the helper genome in Cre+ cells,
preventing it from spreading. Cre will also recombine the amplicon, allowing it to express TK such that the helper can replicate and facilitate production of tracer amplicons that spread to and express GFP in downstream neurons. Cre-negative neurons infected with helper and tracer will initially lack TK and will therefore not be able to produce amplicon tracer. Therefore, the downstream neurons of Cre-neurons would not be labeled. Note however, that for brain regions in which Cre-positive and Cre-negative neurons are interconnected (such as auditory cortex), labeling of downstream neurons will not actually be selective for those connected to the Cre-expressing neurons, as intended. This is because TK expressing amplicons will spread to Cre-negative neurons allowing the helper to be active and produce tracer amplicons in the Cre-negative neurons. See further below.

1: For tracing system 1, it is claimed that helper virus does not spread from the initially infected neurons and that, as a consequence, the spread of tracer is monosynaptic (not multi-synaptic spread). The validity of this claim relies on the unproven assumption that
Cre-recombination from the tracer will successfully recombine and remove pac from every copy of the helper genome. This seems unlikely given that the helper will rapidly replicate in the presence of TK expressed from the tracer. (1) To support these claims it is necessary to show that there is no TdTomato expressed at postsynaptic target regions at very short times (1 or 2 days) after injection of the tracer system. For example, following injections of the tracing system into AC, please show photographs of the MG at d1 and d2 post-injection. Fig. 2d which illustrates that red fluorescent cells have disappeared from the AC between d1 and d5. (Compare to Fig. 1c.) Reply 1-(1): We agree that this is an important concern. As we noted in the revised Methods and manuscript, Cre-excision efficiency is very high -but we acknowledge the possibility that not every pac sequence will be removed from every copy of the helper genome. In anticipation of this potential issue, a very small fraction of helper was also packaged during H129Amp tracer production in Vero cells, resulting in the raw H129Amp tracer product as a mixture of tracer (~95%) and helper (~5%). Upon brain injection of the H129Amp-CTG tracer system, potentially incomplete pac excision could cause replication and transmission of the helper in theory. However, we do not observe helper labeled neurons in the downstream target of the injection site at Day 5 ( Supplementary Fig.   4). As suggested by the reviewer, we tested this more comprehensively by examining the injection site (AC) and its innervating regions (represented by MG) at earlier time points after injection but before Day 5 post injection. No tdTomato labeled neurons are observed in postsynaptic target regions at either Day 1 to Day 3 post injection, indicating that the strategy that we employed for H129Amp tracer production in Vero cells is sound ( Reply Fig. 2).

Are there any red cells observed? Photographs at d5 are not adequate because it is clear that all cells infected with helper would have been killed and eliminated at d5. This is clearly shown in
These results indicated that the helper actually did not transmit to any of the downstream nuclei, despite the acknowledged theoretical possibility of helper-replication due to the Cre-recombination efficiency. We do not know entirely why this works, but we hypothesize the following potential reasons may contribute: i) The replication of H129 in neurons in vivo is slower and less efficient than that in the Vero cells used for H129Amp tracer production. It is possible that the Cre-recombinase rapidly expressed by H129Amp efficiently excises pac in every copy of the helper, and thus fully disarms the helper's packaging ability in neurons. ii) The H129Amp tracer carries multiple copies of pac in the pseudo-genome, while the helper has only 1 copy before excision. Therefore, the replicated tracer may hijack the capsid packaging machinery for its own pseudo-genome, and the un-excised helper genome cannot be efficiently packaged under such conditions. Reply Fig. 2: AC tracing results at early time points. Elements of this can be found in the revised Fig. 2c in the manuscript and Supplementary Fig. 4. The H129Amp tracer system (H129Amp-CTG tracer 1.5×10 8 pfu/ml and helper 1.5×10 8 pfu/ml, in 300 nl) was injected into the auditory cortex (AC, AP: -2.80 mm; ML: -4.13 mm; DV: -2.38 mm) of wildtype C57BL/6 mice, and the brains were collected at 1, 2 and 3 days post-injection for imaging. The representative images of the injection site AC and the direct innervating regions, represented by MG are shown. Images with higher magnifications of the boxed areas are presented in the right panels.
(2) If there are red cells in postsynaptic targets of the AC at d1, then it is clear that they could mediate spread of tracer another synaptic step. Please show results that clearly illustrate a lack of spread in cases where it might be expected. For example, the results state: "… but not in further downstream second order connected nuclei (data not shown). These results show that H129Amp tracer anterogradely labels directly connected postsynaptic neurons limited to monosynaptic connections." It is not possible to show something with results that are not shown. Please specifically state what structures were observed that might have potentially been labeled by multisynaptic spread. Such structures should contain the GFP-labeled axon terminals of neurons that were trans-neuronally labeled with the tracer. But they should not contain labeled postsynaptic neurons. Please provide photographs illustrating the presence of axon terminals in these locations without postsynaptic labeling.

Reply 1-(2):
We apologize for not included the control data showing the absence of multisynaptic spread to downstream brain regions. We agree that this is an important control. The requested data is now presented in Supplementary Figure 5. Auditory cortex (AC) directly innervates lateral amygdala (LA) and contralateral AC (Cont. AC), these brain areas in turn project to central amygdaloid nucleus (CeA) and contralateral medial geniculate nucleus (Cont. MG), respectively (Supplementary Figure 5a). After injecting H129Amp-CTG tracer system into the AC of wildtype C57BL/6 mice, we examined CeA and Mont. MG, the representative 2 nd -order innervating nuclei of AC, at Day 5 (Supplementary Figure 5b). Neither green (GFP) nor red (tdT) labeled cell bodies were observed in these regions (Supplementary Figure  5c and d), thus showing that uncontrolled 2 nd order neuronal labeling does not occur. Further, the H129Amp tracer system was tested in Ai14 reporter mice, Cre expressed by H129Amp-CTG could drive long-term and robust expression of Cre-dependent red fluorescence reporter. After injecting H129Amp-CTG tracer system into the AC of Ai14 reporter mice, we examined the representative 2 nd -order innervating nuclei of AC, CeA and Cont.MG,at Day14 (Supplementary Figure 5e ). No red labeled cell bodies were observed in these regions, while a small amount red axon terminals was observed ( Supplementary Figure 5f and g). These results have been added to the revised manuscript as Supplementary Fig. 5. Reply 2-(1): We appreciate the reviewer raising this essential concern about the potential retrograde labeling of H129 tracer. We are indeed aware of this issue and we have taken steps to mitigate this as detailed below. i) The retrograde labeling of H129 tracers can be minimized by optimizing the tracing parameters. Despite the predominant anterograde transneuronal transmission, H129 tracers have the potential to retrogradely label the upstream neurons by invading the axon terminal and retrograde transportation (PMID: 24585022, 31348990). Our lab has also intensively investigated this retrograde labeling, and discussed this issue in our previous publications (PMID: 28499404, 32824837, 3336799, 35012591). According to our published studies, the retrograde labeling of H129 tracer is associated with many experimental parameters, such as tracer titer, injection volume, tracing duration, injecting site, the circuit to be traced, etc. Carefully optimizing these tracing conditions may effectively minimize or even prevent the potential retrograde labeling (PMID: 28499404). We have tested multiple doses of the H129 tracer in multiple brain regions and from these optimization studies, we determined that the ideal dose of the H129 tracer is ~5.0×10 8 pfu/ml, 150-350nl volume. Using this optimized dose, H129 tracer labels no upstream neurons retrogradely for most tested brain nuclei, except for very few retrogradely labelled cells in CA1 (see Supp. Fig. 8 in Zeng et al., 2017, Mol. Degeneration). The H129Amp tracer system applied in the present study are all at a dose of ~1.5×10 8 pfu/ml, 300nl, which is below the threshold ~5.0×10 8 pfu/ml, 350nl.

2: The authors do not consider the known fact that both the helper and the tracer are packaged with envelope proteins that will mediate retrograde infection. The H-129 strain is biased toward anterograde because it is trafficked down the axons of infected neurons. But there is not any mechanism that prevents infection of axon terminals and retrograde spread to the infected cell bodies. (1) As a result, it is expected that injections of the tracing system into cortical locations (such as AC) will result in direct infection of thalamic neurons (such as in MG). This should be clearly visible as
ii) No retrograde labeling was observed in the test tracing using the V1-SC pathway using the H129Amp tracer system. The V1-SC pathway has been well characterized, and all available evidence indicates that it is unidirectional (PMID: 27989459). In the present study, we performed a test tracing using V1-SC pathway to validate the transmission direction of the H129Amp tracer system. H129Amp tracer system was injected into the SC of Ai14 mice with a dose (~1.5×10 8 pfu/ml, 300nl) lower than the above-mentioned threshold (~5.0×10 8 pfu/ml, 350nl). At Day 14, robust red labeled cell bodies were observed in postsynaptic targets of SC ( Supplementary Fig. 6cg), while no labeled cell bodies in upstream region V1 are detected ( Supplementary Fig. 6d-g). These results indicated that the H129Amp tracer system does not retrogradely label the upstream nuclei under the optimized condition.
iii) The retrograde labeling of H129 tracers can be significantly reduced using gKmut, and we are working on further improvements of the H129Amp tracer system. In a recently published paper, we introduced a novel H129 tracer with reduced retrograde labeling (PMID: 35012591). This is achieved by pseudotyping H129 tracer with the mutant gK (gKmut), an envelope glycoprotein of H129 (see Fig. 4 in Yang et al., 2022, Mol. Degeneration). Currently, we are working on replacing the original wildtype gK (gKwt) with the gKmut in the helper genome. We believe the gKmut replacement should also dramatically reduce the unwanted retrograde labeling of the H129Amp tracer system.

Reply 2-(2):
We tested the transmission ability of the tracer (H129Amp-CTG) without adding additional helper. The raw product of H129Amp-CTG tracer was titrated and adjusted to the optimized tracing titer (1.5×10 8 pfu/ml, This is considered as "tracer alone", since a very small fraction of helper "contamination" is inevitable and irremovable in raw H129Amp tracer product). Without additional helper supplementation, the H129Amp-CTG tracer was injected "alone" into the AC of wildtype C57BL/6 mice, and the brains were examined at Day 1 and Day 5. Green (GFP) labeled neurons were clearly observed at the injection site, but not detected in any other examined brain regions, including the upstream and postsynaptic target regions of AC (Supplementary Figure  2a and b). These results indicate that the tracer alone without additional helper is limited to the injection site. Notably, the derived H129Amp tracer is a mixture containing ~5% helper, as we described in the manuscript and methods. It is hardly to obtain pure high titer H129Amp tracer without any helper "contamination" (PMID: 19956558). The titers of the tracer and helper in the raw tracer product mixture can be determined by plaque-forming assays, and then independently propagated helper needs to be added to the raw tracer product to adjust the tracer/helper to the desired final ratio. In Supplementary Figure 1, we tested the spreading ability of tracer/helper ratio of 10:1, 5:1, 1:1, 1:5, and 1:10. H129Amp tracer (represented by H129Amp-CTG) and H129-dTK-T2-pac Flox helper (the only one, shared in all the experiments) were injected into AC of wildtype C57BL/6 mice as the indicated ratio, and the results were observed at Day 5. For tracer/helper ratios of 10:1 and 1:10, no neurons at the postsynaptic nuclei (represented by Cont. AC) were labeled by GFP. When tracer and helper were applied at ratios of 5:1, 1:5, and 1:1, GFP-labeled neurons were observed at Cont. AC, a representative postsynaptic nucleus. Quantification analysis showed that most GFP-labeled neurons were observed in Cont. AC with a 1:1 tracer/helper ratio ( Supplementary Fig. 1b and c). The results indicated that the H129Amp tracer system didn't spread when the absolute ratio between tracer and helper is too big (exceed 5), which is a similar condition to the raw tracer product (tracer ~95% : helper ~5% = 19:1). These results have been added to the revised manuscript as part of the Supplementary Fig. 2. 3. The second "starter-specific" tracing system is unlikely to actually be specific as intended and claimed. This system utilizes a Cre-dependent tracer (H129Amp-DIO-TG) and is intended to trace the monosynaptic outputs selectively from Cre-expressing cells. The authors do not describe the rationale behind how this should work, but apparently they expect that there will not be any expression of TK in cells that do not express Cre. And since Cre-negative cells at the primary injection site are infected with helper but do not express TK, the helper will not replicate in Cre-negative cells. It is also assumed that tracer will not spread locally between Cre-positive and Cre-negative cells. But these assumptions are certainly wrong for the cortical experiment shown. This is because there are direct connections between the Cre-positive and the Cre-negative cortical cells. It is therefore unlikely that the postsynaptic label observed in distant structures reflects selective outputs from the Cre-positive cortical cells. The expected mechanism for non-selective spread is as follows. Following injections of the tracer and helper into the cortex, both vectors infect both Cre+ and Cre-neurons. In the Cre+ neurons, pac is removed from the helper and the tracer expresses TK and GFP. pac is not removed from the helper that is in the Creneurons. Tracer spreads from the Cre+ to connected Cre-neurons where it expresses TK and allows replication of the helper and further production of tracer amplicons in the Crenegative neurons. This allows the tracer to spread an additional synaptic step and to label the outputs of Cre-neurons. In addition, helper with intact pac is also replicated and can spread from the Cre-neurons, resulting in further multisynaptic spread.
The authors do not appear to have considered this and did not use a Cre-expressing mouse line that is capable of demonstrating this artifact. For example, it is expected that if the experiment were conducted in the AC of PV-Cre mice expressing Cre in parvalbumin inhibitory neurons (which make only local cortical connections) "starter-specific" tracing would not result in spread outside the cortex. But the artifact described would allow the spread of tracer from PV interneurons to cortical excitatory neurons, and then from the excitatory cells to postsynaptic neurons in more distant targets. It is necessary to show results from a Cre line in which it is possible for this artifact to be revealed and evaluated. Another such Cre line would be Tlx3-Cre which expresses Cre in cortical layer 5 pyramidal neurons that make cortico-cortical projections but not those that project to subcortical structures. If the spread is actually "starter-specific", use of the system in this line should result in anterograde tracing to cortical targets but no labeling in subcortical structures such as superior colliculus.
Reply 3: We appreciate the reviewer raising this essential concern and offering the experimental strategy to address it. The reviewer describes the possible chain of events that: i) the H129Amp-DIO-TG tracer and helper infect the same Cre + neurons at the injection site; ii) the tracer and helper support each other and new tracer progeny is synthesized, which is not H129Amp-DIO-TG anymore, but consistently expresses TG (similar to H129Amp-TG), because of Cre-induced recombination; iii) this newly synthesized tracer H129Amp-TG may be anterogradely transmitted to local Creneuron innervated by the starter Cre + neuron; iv) if this local Creneuron is also infected by helper, the H129Amp-TG and helper may support each other independently to Cre-recombinase and initiate further anterograde transmission again through this Creneuron. We do agree with the reviewer that if this chain of events were to occur, it would complicate the interpretation for the starter-specific tracing.
To address the reviewer's concern, we performed tracing with H129Amp-DIO-TG tracer system in PV-Cre mice, instead of the Tlx3-Cre mice (which are unavailable for us). PV-Cre mice express Cre recombinase in parvalbumin inhibitory interneurons (making only local cortical connections). The H129Amp-DIO-TG tracer system was injected into the AC of the PV-Cre mice, and the results were examined at Day 5. As shown in Supplementary Fig.11, the green (GFP) labeled neurons are only observed at the injection site, but are not detected in any other brain regions, including known downstream and upstream regions. Thus while the H129Amp-DIO-TG tracer system in theory could initiate the anterograde monosynaptic transmission from a local Creneuron in the chain of hypothetical events described above, this is not detected in practice. The most likely explanation for the absence of Creneuron initiated transmission is that it may be a very low probability event. However, this potential caveat should be taken into account when applying the H129Amp-DIO-TG tracer system for starter-specific tracing. To warn future users of this hypothetical problem, we have added text addressing this in the revised manuscript along with our new control data (lines 491-499, and Supplementary Fig.11).

Reply:
We concur and have amended the revised manuscript to "Although multiple anterograde monosynaptic tracers have been developed based on the herpes simplex virus 1 (HSV-1) strain H129 (H129), functional mapping for output connectome requires further technical development to minimize viral toxicity" (lines 78-81).

"Both retrograde and anterograde monosynaptic tracing are achieved by combining two components: i) a viral tracer (tracer), which is a replication-or transmission-deficient virus
with one or more genes deleted whose trans-neuronally infects connected neurons requires the assistance of ii) a helper virus (helper) which complimentarily expresses the tracer's deficient gene(s), thus supporting tracer replication and transmission." Suggest the following edits: "Both retrograde and anterograde monosynaptic tracing are achieved by combining two components: i) a conditionally competent viral tracer (tracer) and ii) a helper virus (helper). The viral tracer is replication-and/or transmission deficient due to the deletion of one or more genes that are required for transneuronal spread to connected neurons. The helper virus expresses genes which replace or complement tracer's deficient gene(s), thus supporting tracer replication and/or transmission." Reply: We concur and have amended the revised manuscript to "Retrograde and anterograde monosynaptic tracing can be achieved by combining two components: i) a conditionally competent viral tracer (tracer) and ii) a helper virus (helper). Replication-and/or transmission deficiencies due to the deletion of one or more genes that are required for transneuronal spread to connected neurons of the viral tracer component is a feature that enforces monosynaptic spread. The helper virus component expresses genes that complement the tracer's deficient gene(s), thus supporting tracer replication and/or transmission" (lines 81-87).
The following sentence is factually incorrect and needs to be changed. "The AAV helper has limited expression efficiency due to a single copy of target gene and deficiency in replication, thus needs to be administrated first of two separate injections to allow sufficient target expression and accumulation to support replication and transmission of tracer." AAV genomes concatenate to incorporate many gene copies into episomes. Thus, AAV is not limited to a single copy, and is capable of expressing at very high levels. It is more relevant to this manuscript that expression is slow and that AAV has a limited payload. Suggest changing to: "Due to the slow onset of expression for AAV vectors, the use of AAV necessitates two separate injections, first helper and then tracer, to allow sufficient expression and accumulation to support replication and transmission of the tracer." Reply: We concur and have amended the revised manuscript to "Due to the slow onset of expression for AAV vectors, the use of AAV necessitates two separate injections, first helper and then tracer, to allow sufficient expression and accumulation of the complementary gene and to support the tracer's replication and transmission" (lines 90-93 ).
3. The introduction provides no context for the statement: "Moreover, for the first time, input-defined postsynaptic neurons' monosynaptic anterograde tracing is succeeded by combining H129Amp tracer system with H129-dgK-G4 tracer." (1)For the reader to understand this statement it will be necessary to describe the H129-dgK-G4 tracer.
(2)What are the expected advantages (if any) of using H129-dgK-G4 tracer with H129amp tracer? (3)How is it possible to combine these for cell type specific output labeling since H129-dgK-G4 tracer expresses pac and is not disabled by cre-dependent pac removal?
Reply: We can clarify this.
(1) H129-dgK-G4 is an anterograde monosynaptic tracer developed by our laboratory and introduced in a recent publication (PMID: 35012591). It is a gK gene deleted (dgK) recombinant virus with 4 copies of the GFP coding sequence (G4). When infecting neurons alone, H129-dgK-G4 expresses GFP and intensively labels the cells, but it does not spread among neurons due to the lack of gK for virus shedding. In trans expressed gK by AAV complementarily supports H129-dgK-G4 anterograde transmit the downstream target.
(2) Current anterograde tracers achieve monosynaptic 2-order neuronal tracing (A to B), but fail to acheive 3-order tracing. The combination of H129Amp tracer system and H129-dgK-G4 tracer system makes it possible to perform trans-2-synaptic 3-order anterograde tracing (A to B to C).
(3) We assume the reviewer raises this question for the potential application shown in Supplementary Fig. 10, where we have raised 2 different tracing scenarios: A (Cre + ) to B to C, and A to B (Cre + ) to C. Similar to the input-defined output tracing as shown in Fig.4, these cell type-specific tracing examples also apply H129-dgK-G4 and appropriate AAV helper for the second step transmission. In both tracing scenarios, the appropriate H129Amp tracer system was injected into the 1 st -order group of neurons, and then the H129Amp tracer transmits to the 2 nd -order neurons either in a Credependent ( Supplementary Fig. 10a) or -independent ( Supplementary Fig. 10b-c) manner. In the 2 nd -neurons, the H129Amp expresses Flp-recombinase, and initiates the gK expression of the AAV-helper. When H129-dgK-G4 is injected into the 2 nd -order region at Day 21, it propagates upon the assistance of the accumulated gK, and the newly produced H129-dgK-G4 transmitted to and labeled the 3 nd -order neurons. Notably, at the time when H129-dgK-G4 is administrated, the H129Amp transmission to the 2 nd -order neurons has been eliminated as shown in Supplementary Fig. 4. H129Amp and H129-dgK-G4 will not support each other's replication in the 2 nd -order neurons. Therefore, the pac-competence of H129-dgK-G4 is required for successful packaging and transmission for the 2-step tracing.
4. There is nothing in the Introduction or the Results description of generation, production and tracing principles to make it clear that, for the first tracing system described, the initial infection or production of tracer is not cell type specific. Please state clearly that the initial injection of tracer and helper will infect and spread from all types of neurons at the injection site.
Reply: We concur that this could be more clear. As commented by the reviewer, the first H129Amp tracer system (H129Amp-CTG tracer and H129-dTK-T2-pac Flox helper) initiates anterograde transmission from any cell type, and is not capable to perform starter-specific tracing. We have amended the revised manuscript to make this more clear (lines 215-217).

"Abundant AC neurons were labeled by H129Amp tracer (green) and/or helper (red)."
Why are neurons labeled red? Is there a red fluorescent protein expressed from the helper genome? Is the tissue stained with an antibody against the helper? There is nothing in the introduction or the results description of generation, production and tracing principles to indicate that TdTomato is expressed from the helper genome. This can only be guessed by looking at Figure 1b. There is also nothing in the figure 1 legend to indicate that this guess is correct. The following edits are suggested. At line 125 rewrite as: "The H129Amp tracer system is composed of H129Amp tracer and H129-dTK-T2-LoxN-pac-LoxN helper. H129-dTK-T2-LoxN-pac-LoxN helper has the thymidine kinase gene deleted (dTK), expresses two copies of the fluorescent protein tdTomato (T2), and its pac gene can be deleted by Cre-recombination (LoxN-pac-LoxN). Due to the identical infection features of H129amp tracer and helper, the system it requires only a single-injection instead of the conventional sequential twice-injection." Also update the figure 1 legend to clearly indicate what is schematized in 1b.

Reply:
We concur that this could have been more clear and have amended the revised manuscript (lines 140-151). Fig. 2 legend and the figure refer to LP. Based on the photograph it appears that the location indicated by LP is the lateral pulvinar nucleus of the thalamus, which receives direct input from SC. But the figure legend states: "LP, pretectal area;". This is probably wrong.

Reply:
We concur and have corrected this in the revised manuscript ( Supplementary Fig. 6).
7. "These results show that the postsynaptic neurons labeled by H129Amp tracer system maintain normal physiological conditions, thus allowing for functional connection mapping." This result cannot be generalized to all neurons labeled with the tracer. This only applies to cells that are still present after 14 days. Other cells may have died.
Reply: When transmitting to the postsynaptic neurons, H129Amp neither replicates nor expresses any toxic viral proteins, so it doesn't kill cells as other replicable viruses do. However, H129Amp might also induce immune responses due to the carried-over virion proteins and the bacterial DNA sequences in its pseudo-genome. Based on these properties the H129Amp tracer has very low or even no viral toxicity, however we concede that it may still cause mild damage to the cells, possibly due to cell stress or immune responses. We acknowledge this possibility and have amended the text to "Patch clamp analysis of postsynaptic neurons labeled by the H129Amp tracer system indicates that these cells maintain normal physiological conditions and may be thus useful for functional connection mapping. However at present, we cannot rule out possible secondary effects of cell stress or immune responses that may impact yet to be determined aspects of cellular physiology." in the revised manuscript (lines 295-299).
8. "Notably, H129Amp tracer system is also capable of achieving input-defined outputs mapping from specific starter neurons, which cannot be done using transsynaptic AAV2/1." There is no reason to expect that this could not be done with transsynaptic AAV2/1. If AAV2/1 expressing Cre is used in a mouse with Cre-conditional Chr2 expression (either from the genome or from an AAV-DIO-Chr2 injected at the postsynaptic site) then the same experiment could be done.

Reply:
To the best of our knowledge, the AAV2/1 has no cell-specificity at the injection site. That is to say, AAV2/1 cannot perform the output tracing of a neuron, which is the innervating target of the Cre+ neurons. But this can be potentially achieved by the H129Amp tracer as introduced in the manuscript (Supplementary Fig. 8). However, we appreciate the reviewer's comments, and the corresponding expression has been modified to "Notably, H129Amp tracer system is also applicable for input-defined output mapping from specific starter neurons." (lines 306-307).
9. "Fast tracing. All current monosynaptic tracers, both retrograde and anterograde, use AAVs as helpers." This statement is not true. For example, monosynaptic rabies tracing has been conducted using mouse lines that have cre-dependent or tTA-driven expression of the helper genes. Thus, there is no need for injection of AAV helper or to wait for expression of helper genes.